Novel crystalline form of a phosphoric acid salt of a dipeptidyl peptidase-iv inhibitor

ABSTRACT

The present invention relates to a novel crystalline anhydrate polymorph of the dihydrogenphosphate salt of (2R)-4-oxo-4-[3-trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine as well as a process for their preparation, pharmaceutical compositions containing this novel form, and methods of use of the novel form and pharmaceutical compositions for the treatment of diabetes, obesity, and high blood pressure.

FIELD OF THE INVENTION

The present invention relates to a novel crystalline form of adihydrogenphosphate salt of a dipeptidyl peptidase-IV inhibitor. Moreparticularly, the invention relates to a novel crystalline anhydrateForm IV of the dihydrogenphosphate salt of(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine,which is a potent inhibitor of dipeptidyl peptidase-IV (DP-IV). Thisnovel crystalline form of the DP-IV inhibitor is useful for thepreparation of pharmaceutical compositions containing the inhibitorwhich are useful for the treatment and prevention of diseases andconditions for which an inhibitor of dipeptidyl peptidase-IV isindicated, in particular Type 2 diabetes, hyperglycemia, insulinresistance, obesity, and high blood pressure. The invention furtherconcerns pharmaceutical compositions comprising the novel crystallinedihydrogenphosphate salt anhydrate polymorphic Form IV of the presentinvention; processes for preparing the dihydrogenphosphate saltanhydrate Form IV and their pharmaceutical compositions; and methods oftreating conditions for which a DP-IV inhibitor is indicated comprisingadministering a composition of the present invention.

BACKGROUND OF THE INVENTION

Inhibition of dipeptidyl peptidase-Iv (DP-IV), an enzyme thatinactivates both glucose-dependent insulinotropic peptide (GIP) andglucagon-like peptide 1 (GLP-1), represents a novel approach to thetreatment and prevention of Type 2 diabetes, also known as non-insulindependent diabetes mellitus (NIDDM). The therapeutic potential of DP-IVinhibitors for the treatment of Type 2 diabetes has been reviewed: C. F.Deacon and J. J. Holst, “Dipeptidyl peptidase IV inhibition as anapproach to the treatment and prevention of Type 2 diabetes: ahistorical perspective,” Biochem. Biophys. Res. Commun., 294: 1-4(2000); K. Augustyns, et al., “Dipeptidyl peptidase IV inhibitors as newtherapeutic agents for the treatment of Type 2 diabetes,” Exp. Opin.Ther. Patents, 13: 499-510 (2003); and D. J. Drucker, “Therapeuticpotential of dipeptidyl peptidase IV inhibitors for the treatment ofType 2 diabetes,” Exp. Opin. Investig. Drugs, 12: 87-100 (2003).

WO 03/004498 (published 16 Jan. 2003), assigned to Merck & Co.,describes a class of beta-amino tetrahydrotriazolo[4,3-a]pyrazines,which are potent inhibitors of DP-IV and therefore useful for thetreatment of Type 2 diabetes. Specifically disclosed in WO 03/004498 is(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine.

However, there is no disclosure in the above reference of the newlydiscovered crystalline anhydrate Form IV of the dihydrogenphosphate saltof(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amineof structural formula I below (hereinafter referred to as Compound I).

SUMMARY OF THE INVENTION

The present invention is concerned with a novel crystalline anhydrateForm IV of the dihydrogenphosphate salt of the dipeptidyl peptidase-IV(DP-IV) inhibitor(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amineof structural formula I (Compound I). The crystalline anhydrate Form IVof the present invention has advantages in the preparation ofpharmaceutical compositions of the dihydrogenphosphate salt of(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine,such as ease of processing, handling, and dosing. In particular, itexhibits improved physicochemical properties, such as solubility,stability to stress, and rate of solution, rendering it particularlysuitable for the manufacture of various pharmaceutical dosage forms. Theinvention also concerns pharmaceutical compositions containing the novelanhydrate polymorph; processes for the preparation of this anhydrate andits pharmaceutical compositions; and methods for using them for theprevention or treatment of Type 2 diabetes, hyperglycemia, insulinresistance, obesity, and high blood pressure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a characteristic X-ray diffraction pattern of the crystallineanhydrate Form IV of Compound L

FIG. 2 is a carbon-13 cross-polarization magic-angle spinning (CPMAS)nuclear magnetic resonance (NMR) spectrum of the crystalline anhydrateForm IV of Compound L

FIG. 3 is a fluorine-19 magic-angle spinning (MAS) nuclear magneticresonance (NMR) spectrum of the crystalline anhydrate Form IV ofCompound I.

FIG. 4 is a typical DSC curve of the crystalline anhydrate Form IV ofCompound I.

FIG. 5 is a typical thermogravimetric (TG) curve of the crystallineanhydrate Form IV of Compound I.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a novel crystalline anhydrate Form IV of thedihydrogenphosphate salt of (2R)-4-oxo-4-[3(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-12,4,5-trifluorophenyl)butan-2-amineof structural formula I (Compound I):

A further embodiment of the present invention provides the Compound Idrug substance that comprises the crystalline anhydrate Form IV in adetectable amount. By “drug substance” is meant the activepharmaceutical ingredient (API). The amount of crystalline anhydrateForm IV in the drug substance can be quantified by the use of physicalmethods such as X-ray powder diffraction (XRPD), solid-state fluorine-19magic-angle spinning (MAS) nuclear magnetic resonance spectroscopy,solid-state carbon-13 cross-polarization magic-angle spinning (CPMAS)nuclear magnetic resonance spectroscopy, solid state Fourier-transforminfrared spectroscopy, and Raman spectroscopy. In a class of thisembodiment, about 5% to about 100% by weight of the crystallineanhydrate Form IV is present in the drug substance. In a second class ofthis embodiment, about 10% to about 100% by weight of the crystallineanhydrate Form IV is present in the drug substance. In a third class ofthis embodiment, about 25% to about 100% by weight of the crystallineanhydrate Form IV is present in the drug substance. In a fourth class ofthis embodiment, about 50% to about 100% by weight of the crystallineanhydrate Form IV is present in the drug substance. In a fifth class ofthis embodiment, about 75% to about 100% by weight of the crystallineanhydrate Form IV is present in the drug substance. In a sixth class ofthis embodiment, substantially all of the Compound I drug substance isthe crystalline anhydrate Form IV, i.e., the Compound I drug substanceis substantially phase pure anhydrate Form IV.

Another aspect of the present invention provides a method for theprevention or treatment of clinical conditions for which an inhibitor ofDP-IV is indicated, which method comprises administering to a patient inneed of such prevention or treatment a prophylactically ortherapeutically effective amount of the crystalline anhydrate Form IV ofCompound L Such clinical conditions include diabetes, in particular Type2 diabetes, hyperglycemia, insulin resistance, obesity, and high bloodpressure.

The present invention also provides for the use of the crystallineanhydrate Form IV of the present invention in the manufacture of amedicament for the prevention or treatment of clinical conditions forwhich an inhibitor of DP-IV is indicated, in particular, Type 2diabetes, hyperglycemia, insulin resistance, obesity, and high bloodpressure. In one embodiment the clinical condition is Type 2 diabetes.

Another aspect of the present invention provides the crystallineanhydrate Form IV for use in the treatment of clinical conditions forwhich an inhibitor of DP-IV is indicated, in particular, Type 2diabetes, hyperglycemia, insulin resistance, obesity, and high bloodpressure. In one embodiment of this aspect the clinical condition isType 2 diabetes.

The present invention also provides pharmaceutical compositionscomprising the crystalline anhydrate Form IV, in association with one ormore pharmaceutically acceptable carriers or excipients. In oneembodiment the pharmaceutical composition comprises a prophylacticallyor therapeutically effective amount of the active pharmaceuticalingredient (API) in admixture with pharmaceutically acceptableexcipients wherein the API comprises a detectable amount of thecrystalline anhydrate Form IV of the present invention. In a secondembodiment the pharmaceutical composition comprises a prophylacticallyor therapeutically effective amount of the API in admixture withpharmaceutically acceptable excipients wherein the API comprises about5% to about 100% by weight of the crystalline anhydrate Form IV of thepresent invention. In a class of this second embodiment, the API in suchcompositions comprises about 10% to about 100% by weight of thecrystalline anhydrate Form IV. In a second class of this embodiment, theAPI in such compositions comprises about 25% to about 100% by weight ofthe crystalline anhydrate Form IV. In a third class of this embodiment,the API in such compositions comprises about 50% to about 100% by weightof the crystalline anhydrate Form IV. In a fourth class of thisembodiment, the API in such compositions comprises about 75% to about100% by weight of the crystalline anhydrate Form IV. In a fifth class ofthis embodiment, substantially all of the API is the crystallineanhydrate Form IV of Compound I, i.e., the API is substantially phasepure Compound I anhydrate Form IV.

The compositions in accordance with the invention are suitably in unitdosage forms such as tablets, pills, capsules, powders, granules,sterile solutions or suspensions, metered aerosol or liquid sprays,drops, ampoules, auto-injector devices or suppositories. Thecompositions are intended for oral, parenteral, intranasal, sublingual,or rectal administration, or for administration by inhalation orinsufflation. Formulation of the compositions according to the inventioncan conveniently be effected by methods known from the art, for example,as described in Remington's Pharmaceutical Sciences, 17^(th) ed., 1995.

The dosage regimen is selected in accordance with a variety of factorsincluding type, species, age, weight, sex and medical condition of thepatient; the severity of the condition to be treated; the route ofadministration; and the renal and hepatic function of the patient. Anordinarily skilled physician, veterinarian, or clinician can readilydetermine and prescribe the effective amount of the drug required toprevent, counter or arrest the progress of the condition.

Oral dosages of the present invention, when used for the indicatedeffects, will range between about 0.01 mg per kg of body weight per day(mg/kg/day) to about 100 mg/kg/day, preferably 0.01 to 10 mg/kg/day, andmost preferably 0.1 to 5.0 mg/kg/day. For oral administration, thecompositions are preferably provided in the form of tablets containing0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100 and 500milligrams of the API for the symptomatic adjustment of the dosage tothe patient to be treated. A medicament typically contains from about0.01 mg to about 500 mg of the API, preferably, from about 1 mg to about200 mg of API. Intravenously, the most preferred doses will range fromabout 0.1 to about 10 mg/kg/minute during a constant rate infusion.Advantageously, the crystalline anhydrate form of the present inventionmay be administered in a single daily dose, or the total daily dosagemay be administered in divided doses of two, three or four times daily.Furthermore, the crystalline anhydrate form of the present invention canbe administered in intranasal form via topical use of suitableintranasal vehicles, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in theart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen.

In the methods of the present invention, the Compound I anhydrate FormIV herein described in detail can form the APL and is typicallyadministered in admixture with suitable pharmaceutical diluents,excipients or carriers (collectively referred to herein as ‘carrier’materials) suitably selected with respect to the intended form ofadministration, that is, oral tablets, capsules, elixirs, syrups and thelike, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active pharmaceutical ingredient can be combined with anoral, non-toxic, pharmaceutically acceptable, inert carrier such aslactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the oral API can be combinedwith any oral, non-toxic, pharmaceutically acceptable inert carrier suchas ethanol, glycerol, water and the like. Moreover, when desired ornecessary, suitable binders, lubricants, disintegrating agents andcoloring agents can also be incorporated into the mixture. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsinclude, without limitation, starch, methyl cellulose, agar, bentonite,xanthan gum and the like.

The crystalline anhydrate Form IV of Compound I has been found topossess a high solubility in water, rendering it especially amenable tothe preparation of formulations, in particular intranasal andintravenous formulations, which require relatively concentrated aqueoussolutions of the API.

In a still further aspect, the present invention provides a method forthe treatment and/or prevention of clinical conditions for which a DP-IVinhibitor is indicated, which method comprises administering to apatient in need of such prevention or treatment a prophylactically ortherapeutically effective amount of anhydrate Form IV of the presentinvention or a pharmaceutical composition containing a prophylacticallyor therapeutically effective amount of anhydrate Form IV.

The following non-limiting Examples are intended to illustrate thepresent invention and should not be construed as being limitations onthe scope or spirit of the instant invention.

Compounds described herein may exist as tautomers such as keto-enoltautomers. The individual tautomers as well as mixtures thereof areencompassed with compounds of structural formula I.

The term “% enantiomeric excess” (abbreviated “ee”) shall mean the %major enantiomer less the % minor enantiomer. Thus, a 70% enantiomericexcess corresponds to formation of 85% of one enantiomer and 15% of theother. The term “enantiomeric excess” is synonymous with the term“optical purity.”

EXAMPLE

(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminedihydrogenphosphate anhydrate Form IV Preparation of3-trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazinehydrochloride (1-4)

Step A: Preparation of bishydrazide (1-1)

Hydrazine (20.1 g, 35 wt % in water, 0.22 mol) was mixed with 310 mL ofacetonitrile. 31.5 g of ethyl trifluoroacetate (0.22 mol) was added over60 min. The internal temperature was increased to 25° C. from 14° C. Theresulting solution was aged at 22-25° C. for 60 min. The solution wascooled to 7° C. 17.9 g of 50 wt % aqueous NaOH (0.22 mol) and 25.3 g ofchloroacetyl chloride (0.22 mol) were added simultaneously over 130 minat a temperature below 16° C. When the reaction was complete, themixture was vacuum distilled to remove water and ethanol at 27-30° C.and under 26-27 in Hg vacuum. During the distillation, 720 mL ofacetonitrile was added slowly to maintain constant volume (approximately500 mL). The slurry was filtered to remove sodium chloride. The cake wasrinsed with about 100 mL of acetonitrile. Removal of the solventafforded bis-hydrazide 1-1 (43.2 g, 96.5% yield, 94.4 area % pure byHPLC assay).

¹H-NMR (400 MHz, DMSO-d₆): δ 4.2 (s, 2H), 10.7 (s, 1H), and 11.6 (s, 1H)ppm.

¹³C-NMR (100 MHz, DMSO-d₆): δ 41.0, 116.1 (q, J=362 Hz), 155.8 (q, J=50Hz), and 165.4 ppm.

Step B: Preparation of5-(trifluoromethyl)-2-chloromethyl)-1,3,4-oxadiazole (1-2)

Bishydrazide 1-1 from Step A (43.2 g, 0.21 mol) in ACN (82 mL) wascooled to 5° C. Phosphorus oxychloride (32.2 g, 0.21 mol) was added,maintaining the temperature below 10° C. The mixture was heated to 80°C. and aged at this temperature for 24 h until HPLC showed less than 2area % of 1-1. In a separate vessel, 260 mL of IPAc and 250 mL of waterwere mixed and cooled to 0° C. The reaction slurry was charged to thequench keeping the internal temperature below 10° C. After the addition,the mixture was agitated vigorously for 30 min, the temperature wasincreased to room temperature and the aqueous layer was cut. The organiclayer was then washed with 215 mL of water, 215 mL of 5 wt % aqueoussodium bicarbonate and finally 215 mL of 20 wt % aqueous brine solution.HPLC assay yield after work up was 86-92%. Volatiles were removed bydistillation at 75-80 mm Hg, 55° C. to afford an oil which could be useddirectly in Step C without further purification. Otherwise the productcan be purified by distillation to afford 1-2 in 70-80% yield.

¹H-NMR (400 MHz, CDCl₃): δ 4.8 (s, 2H) ppm.

¹³C-NMR (100 MHz, CDCl₃): δ 32.1, 115.8 (q, J=337 Hz), 156.2 (q, J=50Hz), and 164.4 ppm.

Step C: Preparation ofN-[(2Z)-piperazin-2-ylidene]trifluoroacetohydrazide (1-3)

To a solution of ethylenediamine (33.1 g, 0.55 mol) in methanol (150 mL)cooled at −20° C. was added distilled oxadiazole 1-2 from Step B (29.8g, 0.16 mol) while keeping the internal temperature at −20° C. After theaddition was complete, the resulting slurry was aged at −20° C. for 1 h.Ethanol (225 mL) was then charged and the slurry slowly warmed to −5° C.After 60 min at −5° C., the slurry was filtered and washed with ethanol(60 mL) at −5° C. Amidine 1-3 was obtained as a white solid in 72% yield(24.4 g, 99.5 area wt % pure by HPLC).

¹H-NMR (400 MHz, DMSO-d₆): δ 2.9 (t, 2H), 3.2 (t, 2H), 3.6 (s, 2H), and8.3 (b, 1H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆): δ 40.8, 42.0, 43.3, 119.3(q, J=350 Hz), 154.2, and 156.2 (q, J=38 Hz) ppm.

Step D: Preparation of3-trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazinehydrochloride (1-4)

A suspension of amidine 1-3 (27.3 g, 0.13 mol) in 110 mL of methanol waswarmed to 55° C. 37% Hydrochloric acid (11.2 mL, 0.14 mol) was addedover 15 min at this temperature. During the addition, all solidsdissolved resulting in a clear solution. The reaction was aged for 30min. The solution was cooled down to 20° C. and aged at this temperatureuntil a seed bed formed (10 min to 1 h). 300 mL of MTBE was charged at20° C. over 1 h. The resulting slurry was cooled to 2° C., aged for 30min and filtered. Solids were washed with 50 mL of ethanol:MTBE (1:3)and dried under vacuum at 45° C. Yield of triazole 1-4 was 26.7 g (99.5area wt % pure by HPLC).

¹H-NMR (400 MHz, DMSO-d₆): δ 3.6 (t, 2H), 4.4 (t, 2H), 4.6 (s, 2H), and10.6 (b, 2H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆): δ: 39.4, 39.6, 41.0, 118.6(q, J=325 Hz), 142.9 (q, J=50 Hz), and 148.8 ppm.

Step A: Preparation of4-oxo-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one(2-3)

2,4,5-Trifluorophenylacetic acid (2-1) (150 g, 0.789 mol), Meldrum'sacid (125 g, 0.868 mol), and 4-dimethylamino)pyridine (DMAP) (7.7 g,0063 mol) were charged into a 5 L three-neck flask.N,N-Dimethylacetamide (DMAc) (525 mL) was added in one portion at roomtemperature to dissolve the solids. N,N-diisopropylethylamine (282 mL,1.62 mol) was added in one portion at room temperature while maintainingthe temperature below 40° C. Pivaloyl chloride (107 mL, 0.868 mol) wasadded dropwise over 1 to 2 h while maintaining the temperature between 0and 5° C. The reaction mixture was aged at 5° C. for 1 h. Triazolehydrochloride 14 (180 g, 0.789 mol) was added in one portion at 40-50°C. The reaction solution was aged at 70° C. for several h. 5% Aqueoussodium hydrogencarbonate solution (625 mL) was then added dropwise at20-45° C. The batch was seeded and aged at 20-30° C. for 1-2 h. Then anadditional 525 mL of 5% aqueous sodium hydrogencarbonate solution wasadded dropwise over 2-3 h. After aging several h at room temperature,the slurry was cooled to 0-5° C. and aged 1 h before filtering thesolid. The wet cake was displacement-washed with 20% aqueous DMAc (300mL), followed by an additional two batches of 20% aqueous DMAc (400 mL),and finally water (400 mL). The cake was suction-dried at roomtemperature. The isolated yield of final product 2-3 was 89%.

Step B: Preparation of(2Z)-4-oxo-4-[3-trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)but-2-en-2-amine(2-4)

A 5 L round-bottom flask was charged with methanol (100 mL), theketoamide 2-3 (200 g), and ammonium acetate (110.4 g). Methanol (180 mL)and 28% aqueous ammonium hydroxide (58.6 mL) were then added keeping thetemperature below 30° C. during the addition. Additional methanol (100mL) was added to the reaction mixture. The mixture was heated at refluxtemperature and aged for 2 h. The reaction was cooled to roomtemperature and then to about 5° C. in an ice-bath. After 30 min, thesolid was filtered and dried to afford 24 as a solid (180 g); m.p.271.2° C.

Step C: Preparation of(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine(2-5)

Into a 500 ml flask were charged chloro(1,5-cyclooctadiene)rhodium(I)dimer {[Rh(cod)Cl]₂}(292 mg, 1.18 mmol) and (R,S) t-butyl Josiphos (708mg, 1.3 mmol) under a nitrogen atmosphere. Degassed MeOH was then added(200 mL) and the mixture was stirred at room temperature for 1 h. Into a4 L hydrogenator was charged the enamine amide 24 (118 g, 0.29 mol)along with MeOH (1 L). The slurry was degassed. The catalyst solutionwas then transferred to the hydrogenator under nitrogen. After degassingthree times, the enamine amide was hydrogenated under 200 psi hydrogengas at 50° C. for 13 h. Assay yield was determined by HPLC to be 93% andoptical purity to be 94% ee.

The optical purity was further enhanced in the following manner. Themethanol solution from the hydrogenation reaction (18 g in 180 mL MeOH)was concentrated and switched to methyl t-butyl ether (MTBE) (45 mL).Into this solution was added aqueous H₃PO₄ solution (0.5 M, 95 mL).After separation of the layers, 3N NaOH (35 mL) was added to the waterlayer, which was then extracted with MTBE (180 mL+100 mL). The MTBEsolution was concentrated and solvent switched to hot toluene (180 mL,about 75° C.). The hot toluene solution was then allowed to cool to 0°C. slowly (5-10 h). The crystals were isolated by filtration (13 g,yield 72%, 98-99% ee); m.p. 114.1-115.7° C.

¹H NMR (300 MHz, CD₃CN): δ 7.26 (m), 7.08 (m), 4.90 (s), 4.89 (s), 4.14(m), 3.95 (m), 3.40 (m), 2.68 (m), 2.49 (m), 1.40 (bs).

Compound 2-5 exists as amide bond rotamers. Unless indicated, the majorand minor rotamers are grouped together since the carbon-13 signals arenot well resolved:

¹³C NMR (CD₃CN): δ 171.8, 157.4 (ddd, J_(CF)=242.4, 9.2, 2.5 Hz), 152.2(major), 151.8 (minor), 149.3 (ddd; J_(CF)=246.7, 14.2, 12.9 Hz), 147.4(ddd, J_(CF)=241.2, 12.3, 3.7 Hz), 144.2 (q, J_(CF)=38.8 Hz), 124.6(ddd, J_(CF)=18.5, 5.9, 4.0 Hz), 120.4 (dd, J_(CF)=19.1, 6.2 Hz), 119.8(q, J_(CF)=268.9 Hz), 106.2 (dd, J_(CF)=29.5, 20.9 Hz), 50.1, 44.8, 44.3(minor), 43.2 (minor), 42.4, 41.6 (minor), 41.4, 39.6, 38.5 (minor),36.9.

The crystalline free base 2-5 can also be isolated as follows:

-   (a) The reaction mixture upon completion of the hydrogenation step    is charged with 25 wt % of Ecosorb C-941. The mixture is stirred    under nitrogen for one h and then filtered. The cake is washed with    2 L/kg of methanol. Recovery of free base is about 95% and optical    purity about 95% ee.-   (b) The freebase solution in methanol is concentrated to 3.5-4.0    L/kg volume (based on free base charge) and then solvent-switched    into isopropanol (IPA) to final volume of 3.0 L/kg IPA.-   (c) The slurry is heated to 40° C. and aged 1 h at 40° C. and then    cooled to 25° C. over 2 h.-   (d) Heptane (7 L/kg) is charged over 7 h and the slurry stirred for    12 h at 22-25° C. The supernatant concentration before filtering is    10-12 mg/g.-   (e) The slurry is filtered and the solid washed with 30% IPA/heptane    (2 U/kg).-   (f) The solid is dried in a vacuum oven at 40° C.-   (g) The optical purity of the free base is about 99% ee.

The following high-performance liquid chromatographic (HPLC) conditionswere used to determine percent conversion to product: Column: WatersSymmetry C18, 250 mm × 4.6 mm Eluent: Solvent A: 0.1 vol % HClO₄/H₂OSolvent B: acetonitrile Gradient: 0 min 75% A: 25% B 10 min 25% A: 75% B12.5 min 25% A: 75% B 15 min 75% A: 25% B Flow rate: 1 mL/min InjectionVol.: 10 μL UV detection: 210 nm Column temp.: 40° C. Retention times:compound 2-4: 9.1 min compound 2-5: 5.4 min tBu Josiphos: 8.7 min

The following high-performance liquid chromatographic (HPLC) conditionswere used to determine optical purity: Column: Chirapak, AD-H, 250 mm ×4.6 mm Eluent: Solvent A: 0.2 vol. % diethylamine in heptane Solvent B:0.1 vol % diethylamine in ethanol Isochratic Run Time: 18 min Flow rate:0.7 mL/min Injection Vol.: 7 μL UV detection: 268 nm Column temp.: 35°C. Retention times: (R)-amine 2-5: 13.8 min (S)-amine 2-5: 11.2 min

(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifuorophenyl)butan-2-aminedihydrogenphosphate monohydrate

A 250 mL round bottom flask equipped with an overhead stirrer, heatingmantle and thermocouple, was charged with 31.5 mL of isopropanol (IPA),13.5 mL water, 15.0 g (36.9 mmol) of(2R)-4-oxo-4-[3-trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-12,4,5-trifluorophenyl)butan-2-aminefreebase and 4.25 g (36.9 mmol) of 85% aqueous phosphoric acid. Themixture was heated to 75° C. A thick white precipitate formed at lowertemperatures but dissolved upon reaching 75° C. The solution was cooledto 68° C. and then held at that temperature for 2 h. A slurry bed ofsolids formed during this age time [the solution can be seeded with 0.5to 5 wt % of small particle size (alpine milled) monohydrate]. Theslurry was then cooled at a rate of 4° C./h to 21° C. and then heldovernight. 105 mL of IPA was then added to the slurry. After 1 h theslurry was filtered and washed with 45 mL IPA (solids can also be washedwith a water/IPA solution to avoid turnover to other crystal forms). Thesolids were dried on the frit with open to air. 18.6 g of solids wererecovered. The solids were found to greater than 99.8% pure by HPLC areapercentage (HPLC conditions same as those given above). The particlesize distribution analysis of the isolated solids showed a mean PSD of80 microns with 95% less than 180 microns. The crystal form of thesolids was shown to be monohydrate by X-ray powder diffraction andthermogravimetric analysis.

Preparation of(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminedihydrogenphosphate anhydrate Form IV

Form IV was prepared by heating the above monohydrate at 120° C. forabout 2 h or by heating the monohydrate above 58° C. for about 8 h. FormIV is metastable and converts into the crystalline monohydrate slowlyunder ambient conditions and rapidly under high relative humidity (98%)at room temperature. Form IV can also be converted to anhydrate Form Iin about 1 h at a temperature above 140° C.

X-ray powder diffraction studies are widely used to characterizemolecular structures, crystallinity, and polymorphism The X-ray powderdiffraction patterns of the crystalline polymorph of the presentinvention were generated on a Philips Analytical X'Pert PRO X-rayDiffraction System with PW3040/60 console. A PW3373/00 ceramic Cu LEFX-ray tube K-Alpha radiation was used as the source.

FIG. 1 shows the X-ray diffraction pattern for the crystalline anhydrateForm IV. The anhydrate Form IV exhibited characteristic reflectionscorresponding to d-spacings of 17.94, 7.95, and 6.16 angstroms. Theanhydrate Form IV was further characterized by reflections correspondingto d-spacings of 4.65, 4.46, and 4.02 angstroms. The anhydrate Form IVwas even further characterized by reflections corresponding tod-spacings of 5.08, 3.73, and 3.45 angstroms.

In addition to the X-ray powder diffraction pattern described above, thecrystalline anhydrate Form IV of Compound I of the present invention wasfurther characterized by its solid-state carbon-13 and fluorine-19nuclear magnetic resonance (NMR) spectra. The solid-state carbon-13 NMRspectrum was obtained on a Bruker DSX 400WB NMR system using a Bruker 4mm double resonance CPMAS probe. The carbon-13 NMR spectrum utilizedproton/carbon-13 cross-polarization magic-angle spinning withvariable-amplitude cross polarization. The sample was spun at 15.0 kHz,and a total of 1024 scans were collected with a recycle delay of 5seconds. A line broadening of 40 Hz was applied to the spectrum beforeFT was performed. Chemical shifts are reported on the TMS scale usingthe carbonyl carbon of glycine (176.03 p.p.m.) as a secondary reference.

The solid-state fluorine-19 NMR spectrum was obtained on a Bruker DSX400WB NMR system using a Bruker 4 mm CRAMPS probe. The NMR spectrumutilized a simple pulse-acquire pulse program The samples were spun at15.0 kHz, and a total of 128 scans were collected with a recycle delayof 5 seconds. A vespel endcap was utilized to minimize fluorinebackground. A line broadening of 100 Hz was applied to the spectrumbefore FT was performed. Chemical shifts are reported usingpoly(tetrafluoroethylene) (teflon) as an external secondary referencewhich was assigned a chemical shift of −122 p.p.m.

DSC data were acquired using TA Instruments DSC 2910 or equivalentinstrumentation was used. Between 2 and 6 mg sample was weighed into anopen pan. This pan was then crimped and placed at the sample position inthe calorimeter cell. An empty pan was placed at the reference position.The calorimeter cell was closed and a flow of nitrogen was passedthrough the cell. The heating program was set to heat the sample at aheating rate of 10° C./min to a temperature of approximately 250° C. Theheating program was started. When the run was completed, the data wereanalyzed using the DSC analysis program contained in the systemsoftware. The melting endotherm was integrated between baselinetemperature points that are above and below the temperature range overwhich the endotherm was observed. The data reported are the onsettemperature, peak temperature and enthalpy.

Thermogravimetric (TG) data were acquired using a Perkin Elmer model TGA7. Experiments were performed under a flow of nitrogen and using aheating rate of 10° C./min to a maximum temperature of approximately250° C. After automatically taring the balance, 5 to 20 mg of sample wasadded to the platinum pan, the furnace was raised, and the heatingprogram started. Weight/temperature data were collected automatically bythe instrument. Analysis of the results was carried out by selecting theDelta Y function within the instrument software and choosing thetemperatures between which the weight loss was to be calculated. Weightlosses are reported up to the onset of decomposition/evaporation.

FIG. 2 shows the solid-state carbon-13 CPMAS NMR spectrum for thecrystalline anhydrate Form IV of Compound I.

FIG. 3 shows the solid-state fluorine-19 MAS NMR spectrum for thecrystalline anhydrate Form IV of Compound I. Form IV exhibitedcharacteristic signals with chemical shift values of −64.7, −104.5, and−135.6 p.p.m. Further characteristic of Form IV are the signals withchemical shift values of −95.7, −111.3, and −148.2 p.p.m.

FIG. 4 shows the differential calorimetry scan for the crystallineanhydrate Form IV. Form IV exhibited a melting endotherm with an onsettemperature of 211.1° C., a peak temperature of 213.3° C., and anenthalpy of 93.0 J/g.

FIG. 5 shows a characteristic thermogravimetric analysis (TGA) curve forthe crystalline anhydrate Form IV. TGA indicated a weight loss of about0.05% from ambient temperature to about 197° C.

The crystalline Compound I anhydrate Form IV of the present inventionhas a phase purity of at least about 5% of Form IV with the above X-raypowder diffraction, fluorine-19 MAS NMR, carbon-13 CPMAS NMR, and DSCphysical characteristics. In one embodiment the phase purity is at leastabout 10% of Form IV with the above solid-state physicalcharacteristics. In a second embodiment the phase purity is at leastabout 25% of Form IV with the above solid-state physicalcharacteristics. In a third embodiment the phase purity is at leastabout 50% of Form IV with the above solid-state physicalcharacteristics. In a fourth embodiment the phase purity is at leastabout 75% of Form IV with the above solid-state physicalcharacteristics. In a fifth embodiment the phase purity is at leastabout 90% of Form IV with the above solid-state physicalcharacteristics. In a sixth embodiment the crystalline Compound I is thesubstantially phase pure Form IV with the above solid-state physicalcharacteristics. By the term “phase purity” is meant the solid statepurity of the Compound I anhydrate Form IV with regard to anotherparticular crystalline or amorphous form of Compound I as determined bythe solid-state physical methods described in the present application.

Examples of Pharmaceutical Compositions:

1) Direct Compression Process:

Compound I anhydrate Form IV (API) is formulated into a tablet by adirect compression process. A 100 mg potency tablet is composed of 124mg of the API, 130 mg microcrystalline cellulose, 130 mg of mannitol (or130 mg of dicalcium phosphate), 8 mg of croscarmellose sodium, 8 mg ofmagnesium stearate and 16 mg of Opadry white (proprietary coatingmaterial made by Colorcon, West Point, Pa.). The API, microcrystallinecellulose, mannitol (or dicalcium phosphate), and croscarmellose sodiumare first blended, and the mixture is then lubricated with magnesiumstearate and pressed into tablets. The tablets are then film coated withOpadry White.

2) Roller Compaction Process:

Compound I anhydrate Form IV is formulated into a tablet by a rollercompaction process. A 100 mg potency tablet is composed of 124 mg of theAPI, 195 mg microcrystalline cellulose, 65 mg of mannitol, 8 mg ofcroscarmellose sodium, 8 mg of magnesium stearate and 16 mg of Opadrywhite (proprietary coating material made by Colorcon, West Point, Pa.).The API, microcrystalline cellulose, mannitol, and croscarmellose sodiumare first blended, and the mixture is then lubricated with one third thetotal amount of magnesium stearate and roller compacted into ribbons.These ribbons are then milled and the resulting granules are lubricatedwith the remaining amount of the magnesium stearate and pressed intotablets. The tablets are then film coated with Opadry White.

1. A dihydrogenphosphate salt of(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amineof structural formula I:

characterized as being a crystalline anhydrate Form IV.
 2. Thecrystalline anhydrate Form IV of claim 1 characterized by characteristicreflections obtained from the X-ray powder diffraction pattern atspectral d-spacings of 17.94, 7.95, and 6.16 angstroms.
 3. Thecrystalline anhydrate Form IV of claim 2 further characterized bycharacteristic reflections obtained from the X-ray powder diffractionpattern at spectral d-spacings of 4.65, 4.46, and 4.02 angstroms.
 4. Thecrystalline anhydrate Form IV of claim 3 further characterized bycharacteristic reflections obtained from the X-ray powder diffractionpattern at spectral d-spacings of 5.08, 3.73, and 3.45 angstroms.
 5. Thecrystalline anhydrate Form IV of claim 4 further characterized by theX-ray powder diffraction pattern of FIG.
 1. 6. The crystalline anhydrateForm IV of claim 1 characterized by a solid-state fluorine-19 MASnuclear magnetic resonance spectrum showing signals at −64.7, −104.5,and −135.6 p.p.m.
 7. The crystalline anhydrate Form IV of claim 6further characterized by a solid-state fluorine-19 MAS nuclear magneticresonance spectrum showing signals at −95.7, −111.3, and −148.2 p.p.m.8. The crystalline anhydrate Form TV of claim 7 further characterized bythe solid-state fluorine-19 MAS nuclear magnetic resonance spectrum ofFIG.
 3. 9. The crystalline anhydrate Form IV of claim 1 characterized bythe solid-state carbon-13 CPMAS nuclear magnetic resonance spectrum ofFIG.
 2. 10. The crystalline anhydrate Form IV of claim 1 characterizedby the thermogravimetric analysis curve of FIG.
 5. 11. The crystallineanhydrate Form IV of claim 1 characterized by the differential scanningcalorimetric (DSC) curve of FIG.
 4. 12. A dihydrogenphosphate salt of(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amineof structural formula I:

comprising a detectable amount of crystalline anhydrate Form IV.
 13. Adihydrogenphosphate salt of(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amineof structural formula I:

comprising substantially all by weight of crystalline anhydrate Form IV.14. A pharmaceutical composition comprising a therapeutically effectiveamount of the salt of claim 1 in association with one or morepharmaceutically acceptable carriers or excipients.
 15. A method oftreating Type 2 diabetes comprising administering to a patient in needof such treatment a therapeutically effective amount of the saltaccording to claim
 1. 16. (canceled)
 17. (canceled)